title: Protein thermal shifts to identify low molecular weight fragments.
abstract: Measuring the strength of binding of low molecular weight ligands to a target protein is a significant challenge to fragment-based drug discovery that must be solved. Thermal shift assays are uniquely suited for this purpose, due to the thermodynamic effects of a ligand on protein thermal stability. We show here how to implement a thermal shift assay, describing the basic features and analysis of the protein unfolding data. We then describe in detail the effects of a ligand on the observed stability of the protein to produce a shift in stability. The anatomy of ligand-induced thermal shift data is discussed in detail. We describe the unique aspects of concentration-response curves, the effect of protein unfolding energetics, and the stoichiometry of the interaction. We outline a typical assay development strategy for optimizing dye type and concentration, protein concentration, and buffer conditions. Guidelines are presented to demonstrate the limits of detection for weak-binding ligands, as applied to sulfonamide-based inhibitors of carbonic anhydrase II and applied to nucleotide binding to the death-associated protein kinase 1 catalytic domain.
authors.affiliation: Biopharmaceutical Technologies, GlaxoSmithKline Biopharmaceutical Research and Development, Upper Merion, Pennsylvania, USA.
authors.forename: James K
authors.initials: JK
authors.lastname: Kranz
authors.forename: Celine
authors.initials: C
authors.lastname: Schalk-Hihi
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors / Q000737:chemistry; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry; D064550:Death-Associated Protein Kinases; D055808:Drug Discovery / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008024:Ligands; D008970:Molecular Weight; D009711:Nucleotides; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry; D013449:Sulfonamides / Q000494:pharmacology; D013816:Thermodynamics
chemical_list: D000817:Anilino Naphthalenesulfonates; D051017:Apoptosis Regulatory Proteins; D002257:Carbonic Anhydrase Inhibitors; D005456:Fluorescent Dyes; D008024:Ligands; D009711:Nucleotides; D011506:Proteins; D013449:Sulfonamides; C027132:1-anilino-8-naphthalenesulfonate; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D024402:Carbonic Anhydrase II